Bead Bound Combinatorial Oligonucleoside Phosphorothioate And Phosphorodithioate Aptamer Libraries

ABSTRACT

The present invention includes composition and methods for making and using a combinatorial library having two or more beads, wherein attached to each bead is a unique nucleic acid aptamer that have disposed thereon a unique sequence. The library aptamers may be attached covalently to the one or more beads, which may be polystyrene beads. The aptamers may include phosphorothioate, phosphorodithioate and/or methylphosphonate linkages and may be single or double stranded DNA, RNA or even PNAs.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of aptamerlibraries, and more particularly, to enhancing availability and use ofaptamers for screening, including high-throughput screening, of primaryor secondary target molecules by using thioated aptamers bound to asubstrate.

BACKGROUND OF THE INVENTION

This application is a continuation in part application based on U.S.patent application Ser. No. 10/272,509, filed Oct. 16, 2002, and acontinuation in part of U.S. Provisional Patent Application No.60/334,887, filed on Nov. 15, 2001. This work was supported by thefollowing United States Government grants DARPA (9624-107 FP), NIH(AI27744) and NIEHS (ES06676). Without limiting the scope of theinvention, its background is described in connection witholigonucleotide agents and with methods for the isolation of sequencesthat are bound by nucleic acid binding molecules and the like.

Virtually all organisms have nuclease enzymes that degrade rapidlyforeign DNA as an important in vivo defense mechanism. The use,therefore, of normal oligonucleotides as diagnostic or therapeuticagents in the presence of most bodily fluids or tissue samples isgenerally precluded. It has been shown, however, thatphosphoromonothioate or phosphorodithioate modifications of the DNAbackbone in oligonucleotides can impart both nuclease resistance andenhance the affinity for target molecules, such as for example thetranscriptional regulating protein NF-κB. Thus, from the foregoing, itis apparent there is a need in the art for methods for generatingaptamers that have enhanced binding affinity for a target molecule, aswell as retained specificity. Also needed are ways to identify andquantify in detail the mechanisms by which aptamers interact with targetmolecules.

Current DNA array technology is problematic in that it is focused on theidentification and quantification of a single mRNA species, and does notprovide information on the more relevant level of functional proteinexpression and in particular protein-protein interactions such asbetween heterodimers and homodimers. Although microarrays have been usedfor detecting the proteome, most of these are based on antibodies ornormal backbone aptamers.

Synthetic phosphodiester-modified oligonucleotides such asphosphorothioate oligonucleotide (S-ODN) and phosphorodithioateoligonucleotide (S₂-ODN) analogues have increased nuclease resistanceand may bind to proteins with enhanced affinity. Unfortunately, ODNspossessing high fractions of phosphorothioate or phosphorodithioatelinkages may lose some of their specificity and are “stickier” towardsproteins in general than normal phosphate esters, an effect oftenattributed to non-specific interactions. The recognition of nucleic acidsequences by proteins involves specific sidechain and backboneinteractions with both the nucleic acid bases as well as the phosphateester backbone, effects which may be disrupted by the non-specificinteractions caused with S-ODN and S₂-ODN analogues.

Gorenstein, et al., U.S. Pat. No. 6,423,493, have taken advantage ofthis “stickiness” to enhance the affinity of S-ODN and S₂-ODN agents fora protein target. A screening method was developed to optimize the totalnumber of phosphorothioate or phosphorodithioate linkages that:decreased non-specific binding to the protein target while enhancingonly the specific favorable interactions with the target protein.

Other advances in combinatorial chemistry allow construction andscreening of large random sequence nucleic acid “aptamer” libraries(e.g., Ellington, A. D. and Szostak, J. W. (1990) In vitro selection ofRNA molecules that bind specific ligands. Nature, 346, 818-822);targeting proteins (e.g., Bock, L. C., et al., (1992) Selection ofsingle-stranded DNA molecules that bind and inhibit human thrombin.Nature, 355, 564-566); and other molecules (Koizumi, M. and Breaker, R.R. (2000) Molecular recognition of cAMP by an RNA aptamer. Biochemistry,39, 8983-8992; Gold, L., et al. (1997) SELEX and the evolution ofgenomes. Curr. Opin. Genetic. Dev., 7, 848-851; and Ye, X., et al.(1996) Deep penetration of an α-helix into the widened RNA major groovein the HIV-1 Rev peptide-RNA aptamer complex. Nat. Struct. Biol., 3,1026-1033).

The identification of specific S-ODN and S₂-ODN aptamers(“thioaptamers”) that bind proteins based upon in vitro combinatorialselection methods, however, is limited to substrates only accepted bypolymerases required for reamplification of selected libraries by thepolymerase chain reaction (PCR). One disadvantage of using thepolymerization of substituted nucleoside 5′-triphosphates into ODNaptamers are the restrictions on the choice of P-chirality by theenzymatic stereospecificity. For example, it is known that[S_(P)]-diastereoisomers of dNTP(αS) in Taq-catalyzed polymerizationsolely yield [R_(P)]-phosphorothioate stereoisomers (Eckstein, F. (1985)Nucleoside phosphorothioates. Annu. Rev. Biochem., 54, 367-402).Therefore, using current methods it is not possible to select[S_(P)]-phosphorothioate stereoisomers along with achiral S₂-ODNanalogous since both [R_(p)]-diastereoisomers of dNTP(αS) and nucleosidedNTP(αS₂) are not substrates of polymerases. Additionally, these invitro combinatorial selection methods require many iterative cycles ofselection and reamplification of the bound remaining members of thelibrary by the PCR, which are quite time consuming.

What is needed are compositions and methods that permit the isolationof, e.g., individual aptamer:protein complexes without the need forrepeated iterative cycles of selection and reamplification of likelybinding targets. Also needed are compositions, methods and systems thatpermit the creation, isolation, sequencing and characterization ofmaking [S_(P)]-phosphorothioate stereoisomers along with achiral S₂-ODNanalogs using, e.g., [Rp]-diastereoisomers of dNTP(αS) and nucleosidedNTP(αS₂). Also needed are methods for creating libraries that permitnot only the isolation of a primary aptamer:protein target, but theisolation of protein(s) that may interact with the aptamer:proteintarget, so called secondary interactions.

SUMMARY OF THE INVENTION

The present invention addressed the problems in the prior art bydeveloping composition and methods for making and using a combinatoriallibrary in which each substrate, e.g., a bead, has attached thereto aunique ODN sequence. More particularly, the one-bead, one-ODN library ofthe present invention includes two or more beads, wherein attached toeach bead is a unique nucleic acid aptamer that have disposed thereon aunique sequence. The bead library aptamers may be attached covalently tothe one or more beads, which may be polystyrene beads. The aptamers mayinclude phosphorothioate, phosphorodithioate and/or methylphosphonatelinkages and may be single or double stranded DNA, RNA or even PNAs.

The ODNs attached to the substrate or bead or the present invention mayalso include one or more predetermined nucleic acid sequences, e.g.,having at least 10, 12, 16, or more bases. The predetermined sequencemay be a 5′ nucleic acid sequence, a 3′ nucleic acid sequence, and a 5′and a 3′ nucleic acid sequence to the ODN. In one embodiment the ODN isattached to, e.g., a polystyrene/polydivinyl benzene copolymer beadwith, e.g., a hexaethyleneglycol linker. The aptamers may be isosteric,isopolar and/or achiral. The aptamers may further include a detectablemarker, e.g., a colorimetric agent such as a fluorophor. The detectablemarker may be attached to the 5′ end, the 3′ end or internally withinthe aptamers. The aptamers of the present invention may be single ordouble stranded.

In another embodiment of the present invention the one-bead, one-ODNcombinatorial library includes two or more beads, attached to each beadis a unique aptamer that has a single unique sequence, and each uniqueaptamer includes a mix of modified and unmodified nucleotides. In oneembodiment the aptamer is double stranded and the modifications to eachstrand is unique and does not mirror the modifications to thecomplementary strand.

Yet another embodiment of the present invention is an ODN library inwhich a library substrate has a surface and attached to the librarysubstrate are the individual beads of a one-bead, one-ODN bead library.The library substrate may be, e.g., a bead, a chip, a chip that includesa capacitance-coupled detector, a photolithographically etched microwellplate to contain beads (“Texas tongue”) or even a glass slide.

The present invention also includes a method of making a combinatoriallibrary including the steps of: attaching a single base to a first beadin a first column and a single thio-modified base (or a differentnucleoside or modified nucleoside monomeric unit) to a second bead in asecond column and mixing the first and second beads. Next, the mixedfirst and second beads are split into the first and second columns, anew base is added to the separated beads in each of the first and secondcolumns and the steps of mixing the beads, splitting the beads andadding a new base in each of the first and second columns are repeateduntil the library is complete. The aptamers may be converted todouble-stranded aptamers using, e.g., a DNA polymerase I Klenowfragment.

Yet another embodiment of the present invention is a method fordetecting nucleic acid-protein interactions by mixing a one-bead,one-ODN combinatorial library with one or more proteins and detectingthe binding of the protein to one or more beads of the one-bead, one-ODNcombinatorial library. The reaction is carried-out generally underconditions that permit the binding of a second protein to the proteinbound to the one or more beads. Also, the sequence on the bead may bedetermined by isolating the bead and sequencing the unique aptamer boundto the bead, which may be done, e.g., to compare the level of proteinbound to one or more of the beads a control and a test sample. The levelof protein bound to the one or more beads may also be from a control anda patient sample.

Another method of the present invention is a way to identify proteinsdifferentially expressed in a sample, by mixing a one-bead, one-ODNaptamer bead library where the sample has been labeled with a first dyeand with a control labeled with a second dye under conditions that allowbinding followed by sorting the bead library and comparing the relativelevels of each of the first and second dyes on each bead. In this assaythe differences in the level of the first or second dye are used todetermine the level of binding of the sample and the control to thebeads in the bead library. The beads may be sorted by a flow cytometeror even manually. For sample evaluation the present method may alsoinclude the step of isolating the one or more beads and determining thecharacteristics of the bound material by SELDI-MS. The first and seconddyes are fluorescent dyes, e.g., cy3 and cy5. The method may alsoinclude placing the library of beads onto a substrate. Using the methodsof the present invention, it is also possible to increase the amount ofdyes or other chromo or fluorophores to achieve sorting of the beads inthe bead library by a variable selection criteria, e.g., a low, mediumand/or high signal selection criteria.

According to one embodiment of the present invention, the modifiednucleotide aptamer can contain a phosphoromonothioate orphosphorodithioate (“phosphorothioates”) and can be selected from thegroup consisting of dATP(αS), dTTP(αS), dCTP(αS) and dGTP(αS). Inanother embodiment of the present invention, no more than three adjacentphosphate sites of the modified nucleotide aptamer are replaced withphosphorothioate groups. In yet another embodiment of the presentinvention, at least a portion of non-adjacent dA, dC, dG, or dTphosphate sites of the modified nucleotide aptamer are replaced withphosphorothioate groups. In yet another embodiment of the presentinvention, all of the non-adjacent dA, dC, dG, or dT phosphate sites ofthe modified nucleotide aptamer are replaced with phosphorothioategroups. In yet another embodiment of the present invention, all of thenon-adjacent dA, dC, dG, and dT phosphate sites of the modifiednucleotide aptamer are replaced with phosphorothioate groups. In stillanother embodiment of the present invention, substantially allnon-adjacent phosphate sites of the modified nucleotide aptamer arereplaced with phosphorothioate groups.

In accordance with another embodiment of the present invention, thetarget molecule or portion thereof is NF-κB. In accordance with anotherembodiment of the present invention, the aptamer is selected to bindNF-κB or constituents thereof and is essentially homologous to thesequences of oligonucleotides that bind NF-κB but one or morenucleotides have at least one thiophosphate or dithiophosphate group. Inyet another embodiment of the present invention, the aptamer is selectedto bind NF-κB or constituents thereof and wherein at least onenucleotide is an achiral thiophosphate or a dithiophosphate. In yetanother embodiment of the present invention, the aptamer is selected tobind NF-κB or constituents thereof and wherein at least one nucleotideis an achiral thiophosphate or a dithiophosphate.

In yet another embodiment of the present invention, between 1 and 6 ofthe phosphate sites of the modified nucleotide aptamer aredithiophosphates. In another embodiment of the present invention, themodified nucleotide aptamer contains 6 dithioate linkages. In oneembodiment of the invention, the detection method is selectedcolorimetric, chemiluminescent, fluorescent, radioactive, massspectrometric, capacitance coupled electrical, Biacor or combinationsthereof. The apparatus of the present invention may further includeaptamer libraries containing multiple different but related members. Inone embodiment of the present invention, the substrate for the libraryis selected from the group consisting of beads, membranes, glass, andcombinations thereof. The substrate may even be a microarray of beads orother substrates.

In one embodiment of the present invention, an apparatus for monitoringbiological interactions on the surface of the substrate, e.g., a beadlibrary, is disclosed. The library can include a substrate, a modifiednucleotide aptamer attached to the substrate, and a target protein orportion thereof. The target protein or portion thereof may be complexedwith the modified nucleotide aptamer under conditions sufficient toallow complexation between the aptamer and the target protein or portionthereof The modified nucleotide aptamer may include an oligonucleotidehaving a desired binding efficiency for a target protein or portionthereof.

According to one embodiment of the present invention, the modifiednucleotide aptamer is selected by the steps of: attaching a first baseto a bead or other substrate; synthesizing a random phosphodiesteroligonucleotide combinatorial library wherein constituentoligonucleotides comprise at least a set of 5′ and 3′ PCR primernucleotide sequences flanking a randomized nucleotide sequence using asplit synthesis method, adding the next base, wherein at least a portionof at least one of the nucleotides in the mix is thiophosphate-modified,to form a partially thiophosphate-modified oligonucleotide combinatoriallibrary, and repeating the steps of adding a base that is eitherthiophosphate-modified of a phosphodiester linked nucleotide iterativelya population of sequences is obtained.

According to one embodiment of the present invention, the steps in whichany number of beads are included in a column that adds athiophosphate-modified base is limited so that no more than threeadjacent phosphate sites of the modified nucleotide aptamer are replacedwith phosphorothioate groups.

The present thioaptamer methodology may also provide a library andmethod of use for identifying aptamers that are improvement overexisting antisense or “decoy” oligonucleotides because of theirstereochemical purity. Chemically synthesized phosphorothioates may be adiastereomeric mixture with 2^(n) stereoisomers with n being the numberof nucleotides in the molecule. These preparations are unsuitable foruse in humans because only a small fraction of the stereoisomers willhave useful activity and the remaining could have potential adverseeffects. In contrast, enzymatically synthesized oligonucleotides arestereochemically pure due to the chirality of polymerase active sites.Inversion of configuration is believed to proceed from R_(p) to S_(p)during incorporation of dNMPαS into the DNA chain. These chiralphosphormonothioates can be incorporated into the complementary strandof duplexes using polymerases and a mix of normal and at least one, butno more than three of dATP(αS), dTTP(αS), dCTP(αS) and dGTP(αS) (orNTP((αS)'s for RNA thioaptamers) as described in (Gorenstein, D. G., etal., U.S. Pat. No. 6,423,493). The present dithiophosphate aptamers arefree from diastereomeric mixtures.

The present inventors have developed chemically synthesizedcombinatorial libraries of unmodified or modified nucleic acids andmethods for using the same, to select rapidly oligonucleotides that bindto target biomolecules, e.g., proteins. The present inventors used asplit synthesis methodology to create one-bead one-S-ODN and one-beadone-S₂-ODN libraries. Binding and selection of specific beads to thetranscription factor NF-κB p50/p50 protein were demonstrated. Sequencingboth the nucleic acid bases and the positions of any3′-O-thioate/dithioate linkages was carried out by using a novelPCR-based identification tag of the selected beads. The use of aPCR-based identification tag allowed the rapid and convenientidentification of S-ODNs or S₂-ODNs that bound to proteins.Phosphorothioate oligonucleotides (S-ODN) or phosphorodithioateoligonucleotide (S₂-ODNs) with sulfurs replacing one or both of thenon-bridging phosphate oxygens were shown to bind to proteins moretightly than unmodified oligonucleotides, and have the potential to beused as diagnostic reagents and therapeutics.

The present invention is a one-bead, one-compound library made by usinga split synthesis method to create an alternative to in vitrocombinatorial selection methods. One-bead library systems have been usedfor organic molecules (Felder, E. R. (1999) Resins, Linkers AndReactions For Solid-Phase Synthesis Of Organic Libraries. In Miertus, S.(ed.), In Combinatorial Chemistry and Technology, Principles, Methodsand Applications. Marcel Dekker, Inc., NY, pp. 35-51); peptides (Lam, K.S., et al., (1991) A new type of synthetic peptide library foridentifying ligand-binding activity. Nature, 354, 82-84; Lam, K. S., etal., (1997) The “one-bead-one-compound” combinatorial library method.Chem. Rev., 97, 411-448; Lam, K. S. (1995) Synthetic peptide libraries.In Molecular Biology and Biotechnology: A Comprehensive Desk Reference.Meyer, R. A. (ed.) p. 880. VCH Publisher: NY.); and oligosaccharidelibraries (Zhu, T., and Boom, G. J. (1998) A two-directional approachfor the solid-phase synthesis of trisaccharide libraries. Angew. Chem.Int. Ed., 37, 1898-1900; Liang, R., et al., (1996) Parallel synthesisand screening of a solid phase carbohydrate library. Science, 274,1520-1522; Hilaire, P. M. St. and Meldal, M. (2000) Glycopeptide andoligosaccharide libraries. Angew. Chem. Int. Ed., 39, 1162-1179). Thepresent invention is the first to demonstrate a one-beadone-oligonucleotide (one-ODN) (e.g., S-ODN, S₂-ODN or RNA) combinatoriallibrary selection methodology used to identifying a specificoligonucleotide aptamer that binds to specific proteins or othermolecules.

Furthermore, the present invention may use S₂-ODN reagents with sulfursreplacing both of the non-bridging phosphate oxygens that are isostericand isopolar with the normal phosphorodiester and are particularlyadvantageous for binding and screening. Importantly, S₂-ODNs are achiralabout the dithiophosphate center, which eliminates problems associatedwith diastereomeric mixtures generally obtained for the chemicallysynthesized S-ODN. The split synthesis approach described herein wasused for the construction of S-ODN, S₂-ODN and RNA bead-basedthioaptamer libraries. For example, specific S-ODNs and S₂-ODNs wereidentified by screening of the libraries against a transcription factorNF-κB p50 or p65 heterodimers. Sequencing of both the nucleic acid basesand the positions of any 3′-O-thioate/dithioate linkages was carried outby using a novel PCR-based identification tag of the selected beads.

The controlled thiolation methodology and the libraries made thereby areapplicable to the design of specific, nuclease resistant aptamers tovirtually any target, but not limited to, amino acids, peptides,polypeptides (proteins), glycoproteins, carbohydrates, nucleotides andderivatives thereof, cofactors, antibiotics, toxins, and small organicmolecules including, dyes, theophylline and dopamine. The nucleaseresistant aptamers may be targeted against viruses, bateria, parasites,neoplastic cells and the like. It is within the scope of this invention,that the instant thioaptamers encompass further modifications toincrease stability and specificity including, for example, disulfidecrosslinking. It is further contemplated and within the scope of thisinvention that the instant thioaptamers encompass further modificationsincluding, for example, radiolabeling and/or conjugation with reportergroups, such as biotin or fluorescein, or other functional or detectablegroups for use in in vitro and in vivo diagnostics and therapeutics.

The present invention further provides the application of thismethodology to the generation of novel thiolated aptamer librariesspecific for nuclear factors such as, for example, NF-IL6 and NF-κB. Bytaking advantage of cognate binding motifs, the library may be focusedto reduce library size, while also taking into account the requireddiversity of ODN species. The NF-κB/Rel family of transcription factorsare key mediators of immune and acute phase responses, apoptosis, cellproliferation and differentiation. The NF-κB/Rel transcription factorsare also key transcriptional regulators acting on a multitude of humanand pathogen genes, including HIV-1.

The present structure-based dithiophosphate and combinatorialmonothiophosphate library system provides for the identification ofaptamers that have high specificity, and high affinity for DNA bindingproteins, for example, a single NF-κB heterodimer, in a cellular extractin a rapid, single well assay followed by, e.g., rapid sorting of thebead using a flow-cytometer calibrated to the bead size(s) used to makethe library. The present invention encompasses the development ofseparate aptamers targeting any one of the 15 possible combinations of,e.g., 5 homo- and hetero-dimers of the 5 different forms of NF-κB/Rel.NF-κB/Rel proteins are not only capable of transactivation (heterodimersthat include NF-κB RelA(p65), c-Rel, RelA, but also repression(homodimers of NF-κB p50 or p52).

The one-bead, one-ODN libraries of the present invention may be used tostudy and in treatment of the many diseases in which transcriptionfactors play a critical role in gene activation, especially acute phaseresponse and inflammatory response. These diseases include, but are notlimited to: bacterial pathogenesis (toxic shock, sepsis), rheumatoidarthritis, Crohn's disease, generalized inflammatory bowel disease,hemorrhagic fevers, autoimmune disorders, asthma, cardio-pulmonarydisease, artherosclerosis, asbestos lung diseases, Hodgkin's disease,prostrate cancer, ventilator induced lung injury, general cancer, AIDS,human cutaneous T cell lymphoma, lymphoid malignancies, HTLV-1 inducedadult T-cell leukemia, atherosclerosis, cytomegalovirus, herpes simplexvirus, JCV, SV-40, rhinovirus, influenza, neurological disorders andlymphomas.

Single-stranded nucleic acids are also known to exhibit uniquestructures. The best documented single-stranded nucleic acid structuresare single-stranded RNA. Single-stranded DNA can also adopt uniquestructures. The present invention is applicable to the selection ofsingle-stranded phosphorothioate aptamers of either RNA or DNA. Suchsingle-stranded aptamers are applicable to both DNA (i.e., cell surfacereceptors, cytokines, etc.) and non-DNA binding proteins.

It is contemplated that the present methods and procedures may bescaled-up as would be necessary for high throughput thioaptamerscreening and selection. For example, 6, 12, 48, 96 and 384 wellmicrotiter plates may be used to select pools of aptamers in theone-bead, one-ODN library to a number of different proteins undernumerous conditions, e.g., for use with in conjunction with a platereader or even an ELISA assay.

According to one embodiment of the present invention, the one-bead,one-ODN library may be employed that discriminates among 100's or even1000's of proteins and particularly protein•protein complexes in thecell, simultaneously. Although the rate of dissociation andequilibration may vary, the rate of dissociation and equilibration ofthe different complexes typically is slow relative to the assay time,which is not a problem for NF-κB/Rel.

An ODN library that includes a substrate for a library, the substratehaving at least one surface. Attached to the surface of the library, isa library of one-bead, one-combinatorial library ODN beads attached tothe library substrate surface, thus making a library of libraries. Thesubstrate may be, e.g., a chip, glass, glass slide, quartz, a goldsurface, a surface plasmon resonance detector, a photolithographicallyetched micromachined microwell chip or “Texas tongue” and the like. Thesubstrate may even be a capacitance coupled detector or other likeelectromagnetic, magnetic, electrical or optical detector. In oneembodiment, the ODN library or libraries is made by attaching a singlebase to a first set of beads in a first column and attaching a mixtureof unmodified or modified nucleotides, which as used herein includesunmodified bases with modified phosphate backbone(s) (i.e., sugarphosphate analogs) to a second set of beads in a second column. Next,the first and second set of beads from the first and second columns aremixed and then again into the first and second columns. A new base or amixture of unmodified or modified bases or phosphate backbone analogsare added to the mixed beads in each of the first and second columns.These steps are repeated until the library is complete. In the finallibrary of libraries, each of the oligonucleotides on each bead is now acombinatorial library and each unique oligonucleotide on each bead mayinclude unmodified or a mix of modified and unmodified nucleotides.

The present invention allows for the identification, isolation andcharacterization of target-specific aptamers and thioaptamers bydispersing the aptamer/thioaptamer combinatorial bead library into a 2Dmatrix. The system and methods of the present invention may use aptamersand/or thioaptamers to target specifically a target, e.g., a peptide,protein, nucleic acid, carbohydrate, lipid or combinations thereof. Thematrix may be a solid matrix or even a thixotrophic matrix. Afterdispersal, the beads in the combinatorial library are identified andselected from within the matrix by matrix mapping and/or chemicalmanipulation of bead spots by a robotic, scanning spot-picker. Availabletechnology, such as the Oncosis Photosis technology provides high speedoptical scanning to image cells and then destroys candidate cells bylaser photolysis. Unlike that technology, the present invention allowsfor the selection, isolation and characterization of specific aptamersand thioaptamers by detecting a signal (or lack thereof) andtransferring bead molecules for subsequent manipulation and/or analysis,e.g., sequencing or characterization of the aptamer and/or the targetusing, e.g., mass spectrometer (MS) instrumentation.

The present inventors had developed a method and system that includesthe steps of: (1) incubating the combinatorial bead library withpurified, labeled protein (target) mixtures or even whole proteosomes;(2) sorting the beads by flow cytometry so as to isolate those beadswhich have bound to protein, utilizing luminescent and other means ofdetection (e.g. fluorescent dye labels, binding to a primary antibodyfollowed by binding to a fluorescent labeled secondary antibody, FRET,chemiluminescence labels, etc.); (3) mass spectrometric (MS) detectionof the protein(s) bound to a single bead; (4) amplification bypolymerase chain reaction (PCR) of the thioaptamer of the protein-boundbead followed by (5) sequencing of that thioaptamer.

The method for aptamer selection of the present invention may alsoinclude the steps of; dispersing a one-aptamer, one-bead combinatorialbead library into a two-dimensional matrix; scanning for aptamer beadsthat generate a detectable signal from interaction between the one ormore aptamer beads and a target; and picking one or more aptamer beadsbased on the detectable signal from within the matrix. The method mayalso includes the step of extracting the target from the aptamer beadand/or the step of identifying the target by mass spectrometry afterliquid chromatography. The one-aptamer, one-bead combinatorial beadlibrary may be dispered within the matrix by molecular printing, e.g.,using an inkjet printer. Examples of the matrix include a gel, apolymer, a thixotropic agent, a glass or a silicon matrix. The methodmay also include the step of separating the target into one or morepeptides prior to separation by liquid chromatography. The step ofidentifying the target by mass spectrometry may be preceded by the stepsof extracting and separating the proteins by liquid chromatography. Inone embodiment, the steps of identifying the target using massspectrometry may be matrix assisted laser desorption ionization (MALDI)mass spectrometry.

Aptamer beads for use with the invention may be, e.g., an S-ODN libraryor an S₂-ODN library. The aptamer portion of the aptamer bead may alsoinclude a colorimetric agent or fluorophore, e.g., one or morefluorophors attached to the 5′ end, the 3′ end or internally within theaptamers. The aptamer may also include a complementary strand to theaptamer. In some embodiments, the aptamer may be a thioaptamer, which isan aptamer that has one or more but less than all of the linkagescomprising one or more of the following: rATP(αS), rUTP(αS), rGTP(αS),rCTP(αS), rATP(αS₂), rUTP(αS₂), rGTP(αS₂), rCTP(αS₂), rATP(αS),dTTP(αS), dGTP(αS), dCTP(αS), dATP(αS₂), dTTP(αS₂), dGTP(αS₂) anddCTP(αS₂), depending on whether the aptamer is an RNA and/or DNAaptamer. The aptamer, the bead and/or the target may be labeled with anenzyme, a dye, a radioisotope, an electron dense particle, a magneticparticle, a fluorescent agent, an antibody, a magnetic particle or achromophore. When the aptamer, bead and/or target are labeled, these maybe detectable with an enzyme, a radioisotope, an electron denseparticle, a magnetic particle, a fluorescent agent, an antibody, amagnetic particle or a chromophore.

The aptamer bead may be further processed to remove the target bound tothe aptamer bead, e.g., when the aptamer bead is acquired by a scanningrobotic head and the target is extracted from the aptamer bead in situ.In one embodiment, e.g., the aptamer bead is acquired by a scanningrobotic head and the target is extracted from the aptamer bead in situby proteolysis and transferred to the inlet of an LC-MS or an LC-MS/MS.Alternatively, the aptamer bead is acquired by a scanning robotic headand the target is extracted from the aptamer bead in situ for MALDI-MSanalysis, wherein the MALDI-MS analysis is selected from the groupconsisting of MALDI-TOF/MS, MALDI-TOF/TOF-MS and MALDI-Q-TOF-MS. Yetother alternatives for the scanning robotic head to acquire the aptamerbead and the target may be: (1) extracted from the aptamer bead in situfor LC-MS analysis, (2) extracted from the aptamer bead in situ forMALDI-MS analysis; and/or (3) extracted from the aptamer bead in situfor MALDI-MS analysis by SELDI ionization. The aptamer bead may befurther processed to remove the target bound to the aptamer bead andanalyzing the target by MS, MS/MS, MALDI-TOF, MALDI-TOF-MS, directsequencing, in some cases the MALDI ionization step may be a SELDIionization. The aptamer bead may also be processed to remove the targetbound to the aptamer bead and the target further analyzed by binding asecond detectable label to the target.

To disperse the aptamer beads before or after being exposed to a targetunder conditions that permit binding, the same may be dispersed,printed, attached or placed into a generally two-dimensional and eventhree-dimensional thixotrophic agent, e.g., a polyacrylamide gel, analkyd resin or a silica-lipid. Following dispersal and binding of theaptamer-bead to the target to form a complex, the complex is imaged andthe beads selected for capture may be selected by picking the beadsmanually, semi-manually or non-manually. Examples of targets include,e.g., peptides, proteins, nucleic acids, carbohydrates, lipids orcombinations thereof. In one example, the aptamer beads may be dispersedwithin the thixotropic agent by molecular printing, e.g., an ink-jetprinter.

Yet another example of the present invention is a system and method foraptamer selection that includes dispersing a one-aptamer, one-beadcombinatorial bead library into a two-dimensional matrix; scanning foraptamer beads that generate a detectable signal from interaction betweenthe one or more aptamer beads and a target; and picking one or moreaptamer beads based on the detectable signal from within the matrix. Theone-aptamer, one-bead combinatorial bead library is dispered within thematrix by molecular printing, e.g., using an inkjet printer. Thetwo-dimensional matrix may be, e.g., a gel, a polymer, a thixotropicagent, a glass or a silicon matrix.

The present invention also includes a system for aptamer selection inwhich a two-dimensional matrix is used to separate two or more aptamerbeads bound to a target; a scanner that images a signal from the two ormore aptamer beads bound to a target; and a spot-picker that picks oneor more aptamer beads bound to a target. The spot-picker is used totransfer one or more aptamer-bead bound to a target to a chamber forfurther chemical manipulation, e.g., aptamer sequencing. The spot-pickermay be a robotic spot picker. The two-dimensional matrix may be a gel, apolymer, a thixotropic agent, a glass or a silicon matrix. In onespecific embodiment, the spot-picker transfers one or more aptamer-beadbound targets to a bead-target separator; and a conduit connected to thechamber allows transfer of the targets into a liquid chromatograph oreven a liquid chromatograph and a mass spectrometer, e.g., a matrixassisted laser desorption ionization mass spectrometer.

The present invention uses a two dimensional (2D) robotic spot picker toidentify a target, e.g., a protein, on an individual bead of acombinatorial bead library that had been incubated with a target to foran aptamer-bead-target complex. The subject invention may use, e.g., acombinatorial bead library/protein incubation step of the existingmethodology. One advantage of the present invention is that it does notrequire flow cytometry to isolate those beads that have bound targetprotein, often a bottleneck for high-throughput analysis. Instead, theinvention scans and picks target bead spots, e.g., a pre-incubatedtarget and aptamer bead library in a polyacrylamide gel, which is thencast onto a 2D plate whose bead “spots” are then imaged, analyzed andmanipulated using a robotic scanning spot picker. The target and theaptamer-beads may be pre-incubated, or the target and/or theaptamer-beads may be incubated in situ. The aptamer-beads and the targetare generally distributed uniformly throughout the two-dimensionalmatrix or agent, e.g., a gel, a gel plate, a glass plate into, e.g., asingle layer. In one example, the aptamer-beads are 20-80 microns indiameter, but may be larger or smaller depending on factors such as thenecessary signal-to-noise ration, amount of sample, number of beads inan aptamer-bead library, etc.

To capture, scan, image, identify and locate the spots to be picked,software available with robotic scanners and pickers, known in the art,is used to determine the XY coordinates and intensity of each bead inthe 2D gel matrix, and directs and controls the hardware formanipulation of aptamer-beads and their target. For example, a highspeed optical scanner based on a positional scanning excitation source(e.g. laser, diode laser) and an emission sensor (e.g. CCD camera) maybe used to create a 2D image/map of candidate beads. Examples ofinstruments for use with the present invention include commerciallyavailable robotic spot pickers, e.g., a Bruker Daltonics Proteineer spIIspot-picker (Bruker Daltonics website). Such spot pickers may beoperated in conjunction with a 2D matrix, e.g., an polyacrylamideelectrophoretic gel, performing 2D gel imaging, detection of proteinspots of the gel, software selection, directing the robotic head to cutout (“core”) a relevant spot (either automatically or based on manualreview of the image map) and transfer of the spot(s) to a sample arrayfor MS analysis. An example of a 2D imaging system is availablecommercially from Oncosis (Oncosis website), which uses “Photosis”technology (WO 98/42356, 1998; WO 01/40454, 2001). The Oncosis system iscapable of high speed laser-based optical scanning to image a 2D matrixof cells embedded in a polymeric matrix, however, the system thenre-addresses and destroys cells that are not of interest via highintensity laser photolysis.

The following are general requirements of a coring mechanism for usewith the robotic head of the spot picker for use with the presentinvention. The coring mechanism of the robotic head of the spot picker,will generally transfer a candidate bead from the cast gel matrix to,e.g., a mass spectrometer inlet. To do so it will generally capture onlyone bead at a time. As such, the coring mechanism will generally bedesigned to have a diameter less than that of two beads. To introduce abead into the interior of a cylinder, e.g., a needle, the inner diameterof the cylinder has to be greater than that of one bead. However, if abead or plug is not to be fully captured within the cylinder, then theitem may be held outside the tip of the cylinder using, e.g.,application of a vacuum to the other end of the cylinder to cause avacuum within the cylinder. Thus, there is a suitable range between onebead diameter and less than two bead diameters.

For example, if the beads are 60 micron diameter, the coring mechanismmust have a diameter between 80-100 microns. If this requirement is notmet, the possibility exists that the coring mechanism will transfer morethan one bead in a single “core.” The “bystander” bead(s) transferredmay also be candidate beads (also bind target protein and thus haveappropriate fluorescent signature) or may be non-candidates (do not bindprotein, do not have fluorescent signature). Where the single “core”contains “bystander” candidate beads, the mass spectrum of the multipleproteins (or peptide digests if on-gel digestion is used) will bedifficult to interpret. Where the single “core” contains “bystander”non-candidate beads, the identification of the binding thioaptamer ofthe candidate bead will be difficult.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIG. 1 shows a MALDI (SELDI) identification of proteins bound to aptamerbeads by either direct laser desorption and MALDI MS detection of p50NF-κB or dissociation of protein from beads and MALDI detection;

FIG. 2 is a schematic diagram for split synthesis of thioaptamer beadcombinatorial libraries or library of libraries;

FIG. 3 shows a fluorigenic sandwich assay for identifying proteins boundto thioaptamer beads;

FIG. 4 shows a sandwich assay demonstrating two color imaging of eitherp50 or p65 forms of NF-κB bound to aptamer beads;

FIG. 5 shows the SELDI (MALDI) identification of a 105 kDa protein innuclear extracts form macrophages activated by LPS, bound to thioaptamersurfaces (ProteinChip);

FIG. 6 shows the identification of thioaptamer sequences selected forhighest binding to NF-κB using either direct flurorescence labeling ofprotein or sandwich assay using “one bead PCR” method);

FIG. 7 is a drawing that summarized a selection scheme of the subjectinvention;

FIG. 8 is a photomicrograph of gel without beads;

FIG. 9 is a photomicrograph of gel in which was dispersed 50 ul offluorescent-labeled beads (approximately 1.4×10⁴ beads;

FIG. 10 is a photomicrograph of gel in which was dispersed 100 ul offluorescent-labeled beads (approximately 3×10⁴ beads); and

FIG. 11 is a photomicrograph of gel in which was dispersed 150 ul offluorescent-labeled beads (approximately 4.2×10⁴ beads).

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

As used herein, “synthesizing” of a random combinatorial library refersto chemical methods known in the art of generating a desired sequence ofnucleotides including where the desired sequence is random. Typically inthe art, such sequences are produced in automated DNA synthesizersprogrammed to the desired sequence. Such programming can includecombinations of defined sequences and random nucleotides.

“Random combinatorial oligonucleotide library” means a large number ofoligonucleotides of different sequence where the insertion of a givenbase at given place in the sequence is random. “PCR primer nucleotidesequence” refers to a defined sequence of nucleotides forming anoligonucleotide which is used to anneal to a homologous or closelyrelated sequence in order form the double strand required to initiateelongation using a polymerase enzyme. “Amplifying” means duplicating asequence one or more times. Relative to a library, amplifying refers toen masse duplication of at least a majority of individual members of thelibrary.

As used herein, “thiophosphate” or “phosphorothioate” are usedinterchangeably to refer analogues of DNA or RNA having sulphur in placeof one or more of the non-bridging oxygens bound to the phosphorus.Monothiophosphates or phosphoromonothioates [αS] have only one sulfurand are thus chiral around the phosphorus center. Dithiophosphates aresubstituted at both oxygens and are thus achiral. Phosphoromonothioatenucleotides are commercially available or can be synthesized by severaldifferent methods known in the art. Chemistry for synthesis of thephosphorodithioates has been developed by one of the present inventorsas set forth in U.S. Pat. No. 5,218,088 (issued to Gorenstein, D. G. andFarschtschi, N., Jun. 8, 1993 for a Process for PreparingDithiophosphate Oligonucleotide Analogs via NucleosideThiophosphoramidite Intermediates), relevant portions incorporatedherein by reference.

“Modified” is used herein to describe oligonucleotides or libraries inwhich one or more of the four constituent nucleotide bases of anoligonucleotide are analogues or esters of nucleotides normallycomprising DNA or RNA backbones and wherein such modification confersincreased nuclease resistance. Thiophosphate nucleotides are an exampleof modified nucleotides. “Phosphodiester oligonucleotide” means achemically normal (unmodified) RNA or DNA oligonucleotide. Amplifying“enzymatically” refers to duplication of the oligonucleotide using anucleotide polymerase enzyme such as DNA or RNA polymerase. Whereamplification employs repetitive cycles of duplication such as using the“polymerase chain reaction”, the polymerase may be, e.g., a heat stablepolymerase, e.g., of Thermus aquaticus or other such polymerases,whether heat stable or not.

“Contacting” in the context of target selection means incubating aoligonucleotide library with target molecules. “Target molecule” meansany molecule to which specific aptamer selection is desired.“Essentially homologous” means containing at least either the identifiedsequence or the identified sequence with one nucleotide substitution.“Isolating” in the context of target selection means separation ofoligonucleotide/target complexes, preferably DNA/protein complexes,under conditions in which weak binding oligonucleotides are eliminated.

By “split synthesis” it is meant that each unique member of thecombinatorial library is attached to a separate support bead on a twocolumn DNA synthesizer, a different thiophosphoramidite orphosphoramidite is first added onto both identical supports (at theappropriate sequence position) on each column. After the normal cycle ofoxidation (or sulfurization) and blocking (which introduces thephosphate, monothiophosphate or dithiophosphate linkage at thisposition), the support beads are removed from the columns, mixedtogether and the mixture reintroduced into both columns. Synthesis mayproceed with further iterations of mixing or with distinct nucleotideaddition.

Aptamers may be defined as nucleic acid molecules that have beenselected from random or unmodified oligonucleotides (“ODN”) libraries bytheir ability to bind to specific targets or “ligands.” An iterativeprocess of in vitro selection may be used to enrich the library forspecies with high affinity to the target. The iterative process involvesrepetitive cycles of incubation of the library with a desired target,separation of free oligonucleotides from those bound to the target andamplification of the bound ODN subset using the polymerase chainreaction (“PCR”). The penultimate result is a sub-population ofsequences having high affinity for the target. The sub-population maythen be subcloned to sample and preserve the selected DNA sequences.These “lead compounds” are studied in further detail to elucidate themechanism of interaction with the target.

The present inventors recognized that it is not possible to simplysubstitute thiophosphates in a sequence that was selected for bindingwith a normal phosphate ester backbone oligonucleotide. Simplesubstitution was not practicable because the thiophosphates cansignificantly decrease (or increase) the specificity and/or affinity ofthe selected ligand for the target. It was also recognized thatthiosubstitution leads to a dramatic change in the structure of theaptamer and hence alters its overall binding affinity. The sequencesthat were thioselected according to the present methodology, using asexamples of DNA binding proteins both NF-IL6 and NF-κB, were differentfrom those obtained by normal phosphate ester combinatorial selection.

The present invention takes advantage of the “stickiness” of thio- anddithio-phosphate ODN agents to enhance the affinity and specificity to atarget molecule. In a significant improvement over existing technology,the method of selection concurrently controls and optimizes the totalnumber of thiolated phosphates to decrease non-specific binding tonon-target proteins and to enhance only the specific favorableinteractions with the target. The present invention permits control overphosphates that are to be thio-substituted in a specific DNA sequence,thereby permitting the selective development of aptamers that have thecombined attributes of affinity, specificity and nuclease resistance.

In one embodiment of the present invention, a method of post-selectionaptamer modification is provided in which the therapeutic potential ofthe aptamer is improved by selective substitution of modifiednucleotides into the aptamer oligonucleotide sequence. An isolated andpurified target binding aptamer is identified and the nucleotide basesequence determined. Modified achiral nucleotides are substituted forone or more selected nucleotides in the sequence. In one embodiment, thesubstitution is obtained by chemical synthesis using dithiophosphatenucleotides. The resulting aptamers have the same nucleotide basesequence as the original aptamer but, by virtue of the inclusion ofmodified nucleotides into selected locations in the sequences, improvednuclease resistance and affinity is obtained.

EXAMPLE 1

S-ODN, S₂-ODN and monothio-RNA Split and Pool Synthesis

A split and pool synthesis combinatorial chemistry method was developedfor creating combinatorial S-ODN, S₂-ODN and monothio-RNA libraries (andreadily extended to unmodified ODNs-whether single strand or duplex). Inthis procedure each unique member of the combinatorial library wasattached to a separate support bead. Targets that bind tightly to only afew of the potentially millions of different support beads can beselected by binding the targets to the beads and then identifying whichbeads have bound target by staining and imaging techniques. Themethodology of the present invention allowed the rapid screening andidentification of aptamers that bind to proteins such as NF-κB using anovel PCR-based identification tag of the selected bead.

The dA, dG, dC and dT phosphoramidites were purchased from AppliedBiosystems (Palo Alto, Calif.) or Glen Research (Sterling, Va.). TheBeaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide) was from GlenResearch. The Taq polymerase kits were from Applied Biosystems. The TACloning kit was from Invitrogen. The Klenow DNA polymerase I was fromPromega. Polystyrene beads (60-70 μm) with non-cleavablehexaethyleneglycol linkers with a loading of 36 μmol/g were fromChemGenes Corp (Ashland, Mass.). The Alexa Fluor 488 dye was fromMolecular Probes, Inc (Eugene, Oreg.). The dA, dG, dC and dTthiophosphoramidites were synthesized as previous described (Yang, X-B.,Fennewald, S., Luxon, B. A., Aronson, J., Herzog, N. and Gorenstein, D.G., “Aptamers containing thymidine 3′-O-phosphorodithioates: Synthesisand binding to Nuclear Factor-κB, J. Bioorganic and Medicinal Chemistry,9, 3357-3362 (1999) and refs therein). The ODNs and S—ODNs used in thestudy were synthesized on a 1-μmol scale on an Expedite 8909 System(Applied Biosystems) DNA Synthesizer.

Synthesis of S-ODN and S₂-ODN libraries. Standard phosphoramidite andthiophosphoramidite chemistry was used for the S-ODN and S₂-ODNlibraries, respectively. The libraries were prepared on a 1 μmole scaleof polystyrene beads. The downstream and upstream primers,5′-GGATCCGGTGGTCTG-3′ (SEQ ID NO:1) and 5′-CCTACTCGCGAATTC-3′ (SEQ IDNO:2) were synthesized in parallel on a two-column DNA synthesizer(Expedite 8909, Applied Biosystems). Following the 5′-primer, thesequences programmed on the synthesizer for the combinatorial S-ODNlibrary were 5′-*CA*GT*TG*AG*GG*GA*CT*TT*CC*CA*GG*C-3′ (SEQ ID NO:3) oncolumn 1 and 5′-*cC*tG*cA*cA*tC*tC*aG*gA*tG*aC*tT*t-3′ (SEQ ID NO:4) oncolumn 2. The sequences programmed for the combinatorial S₂-ODN librarywere 5′-ATGT*AGCC*A*GCTAGT*CTG*TCAG-3′ (SEQ ID NO:5) on column 1 and5′-CGCC*cAGT*g*aAGGTG*gaA*CCCC-3′ (SEQ ID NO:6) on column 2. The3′-primer sequence completed the 52-mer programmed on the synthesizer.

A “split and pool” occurred at each position indicated by an asterisk inorder to synthesize the combinatorial region for the S-ODN and S₂-ODN.The lower case letter indicates a 3′-thioate linkage, the upper caseletter indicates a 3′-phosphate linkage, while the lower case boldletter indicates a 3′-dithioate linkage. The coupling yield wastypically upwards of 99% as determined by the dimethoxytrityl cationassay. Sulfurization chemistry used the Beaucage reagent. The fullyprotected S-ODN or S₂-ODN combinatorial libraries with the non-cleavablelinker beads were treated with concentrated ammonia at 37° C. for 21hours to remove the protecting groups while allowing the ODN to remainattached to the beads. The S-ODN or S₂-ODN bead-based single-strand (ss)DNA library was washed with double distilled water. The ssDNA library(typically 1-3 mg of support beads) was converted to a double-strand(ds) DNA by Klenow DNA polymerase I reaction in the presence of DNApolymerase buffer, dNTP mixture and reverse primer according to themanufacture. The dsDNA library was washed twice with phosphate-bufferedsaline (PBS).

Briefly, a solid-phase synthesis of a one-bead one-S/S₂-ODN library isas follows. In a first cycle, in column 1, a phosphoramidite dC was usedto form a dinucleotide phosphotriester dGC via a phosphotriesterlinkage, in column 2, a phosphoramidite T was used to form adinucleotide thiophosphotriester dGt via a phosphothiotriester linkage.Upon pooling, the end products are a mixture of two kinds of bead-bounddinucleotides include phosphorotriester and phosphothiotriester. Aftersplitting and pooling through three such cycles the eight (2³) possibleODN and/or S-ODN tetraoligonucleotides are represented on separatebeads. A lowercase letter denotes a 3′-thioate, while an uppercaseletter denotes a 3′-phosphate. The S₂-ODN library was generated byreplacing the phosphoramidite with a thiophosphoramidite globally incolumn 2. The sulfurization step immediately followed thethiophosphoramidite coupling step.

Labeling NF-κB p50/50 protein with Alexa Fluor 488. To 0.5 ml of p50/50protein (0.215 mg/ml, expressed and purified (King, D., et al. (2002)Combinatorial selection and binding of phosphorothioate aptamerstargeting human NF-κB RelA (p65) and p50. Biochemistry, 41, 9696-9706,relevant portions incorporated herein by reference) in PBS containing30% glycerol was added 50 μl of 1 M bicarbonate. The protein wastransferred to a vial of reactive Alexa Fluor 488 dye and stirred atroom temperature for 1 hr. Fluorescently labeled protein was purifiedaccording to procedures from Molecule Probes, Inc. The labeled proteinwas stored at 4° C. in the dark.

Alexa Fluor 488 labeled NF-κB p50/p50 binding to beads, selection ofbeads. A portion of the ds S-ODN or S₂-ODN library (ca. 3.0 mg of thebeads) was suspended in 300 μl of blocking buffer (PBS containing 0.05%Tween-20) and incubated at room temperature for 1 hr in amicrocentrifuge tube. The beads were washed with 300 μl of PBS andpelleted by centrifugation. The beads were suspended in 300 μl of AlexaFluor 488 labeled NF-κB p50/p50 (0.07 μg/μl) at room temperature for twohrs and then washed with blocking buffer (2×300 μl) and PBS (2×300 μl).A portion of the beads were transferred to a slide and viewed underfluorescent microscope. Individual beads with the highest fluorescenceintensity were removed by a micropipette attached to a micromanipulator,sorted into PCR microcentrifuge tubes and washed with 8M urea (pH 7.2)to remove the bound protein.

One-bead one-PCR amplification and sequencing of PCR product. A selectedsingle bead was mixed with the following PCR components: 6 μl of 25 mMMgCl₂ (8 μl for 15, 10 and 8mer primers), 0.5 μl of Taq polymerase (5units/μl), 1 μl of 8 mM dNTP, and 10 μl of PCR buffer and 1 μl of 40 mMprimers. The PCR was run on a GeneAmp PCR system 2400 (Perkin Elmer).The PCR reaction mixtures were thermal cycled using the following schemefor amplification: 94° C. for 5 min (1 cycle); 94° C. for 2 min, 55° C.for 2 min (35° C. for 10 and 8 mer primers), 72° C. for 2 min (35cycles); and 72° C. for 7 min (1 cycle). The PCR products were analyzedon a 15% native polyacrylamide gel. The PCR product was cloned using theTA Cloning procedure (Invitrogen) and sequenced on an ABI Prism 310Genetic Analyzer (Applied Biosystems).

One-bead one-oligonucleotide libraries. A primary consideration fordesigning a one-bead, one-ODN library using phosphoramidite chemistrywas defining suitable bead linker chemistry where the ODNs can besynthesized and yet remain attached covalently to the beads after fulldeprotection. Additional considerations include development of the splitsynthesis method for construction of the ODN library, screeningbead-based ODN libraries in aqueous media for one-bead binding assaysand sequencing of the ODN bound on the individual bead. Althoughlong-chain alkylamine controlled-pore glass (LCA-CPG) (Pierce ChemicalCo., Rockport, II) has been used for many years for efficient ODNsynthesis, LCA-CPG may not always be suitable for generation ofone-bead, one-ODN libraries. The size, homogeneity and the swelling ofCPG are factors to consider when selecting a chemistry for a one-bead,one-ODN library. For example, one disadvantage of the CPG linkerchemistry available currently is that ODNs are cleaved from the solidsupport during the ammonia deprotection step. An advance in solidsupport chemistry has been the ability to synthesize ODNs on moreuniform polystyrene beads. Importantly, using chemistry with anon-cleavable hexaethyleneglycol linker attaching the firstphosphoramidite (ChemGenes Corp.), the synthesized ODNs are stillattached covalently to the beads after full base and phosphate esterdeprotection. In this procedure each unique ODN chemical entity in thecombinatorial library is attached to a separate support bead. Selectionof a bead-based ODN combinatorial library can then be carried out bybinding the bead library of ODNs to a target protein under highstringency conditions where only a few beads show binding.

Following the in vitro combinatorial selection method for identificationof selected ODN sequences, the ODN sequence on the selected beads may beidentified. For example, 5′ and 3′ fixed ODN primer sequences flankingthe combinatorial library segment of the ODN may be used to aid in theidentification. Fixed primer regions allow PCR amplification of thesequence as well as Klenow extension of the ssDNA attached covalently toproduce a combinatorial library of dsODN attached to the beads.

Primers design for one-bead one-ODN. Initially, a template ODN with apredetermined 14mer sequence region flanked by two 18mer primers on thebeads were synthesized (ODN 1 in Table 1). Its ability to supportone-bead one-PCR amplification was studied for several individual beads.The PCR product was cloned using the TA Cloning procedure and sequencedon an ABI Prism 310 Genetic Analyzer. The desired sequence wasconfirmed. Although 18mer or longer primers are generally used in PCRamplification, shorter primers are attractive since the size of the ODNis limited by the synthesis yields for long ODNs.

TABLE 1 ODNs on beads and primers ODNs on beads used as templates (SEQID NO:7) ODN1:5′-ATGCCTACTCGCGAATTC-CCAGGAGATTCCAC-GGATCCGGTGGTCTGTTC-Bead (SEQ ID NO:8)ODN2:5′-CCTACTCGCGAATTC-AGTTGAGGGGACTTTCCCAGGC- GGATCCGGTGGTCTG-BeadPrimers Upstream primers (SEQ ID NO:9) Downstream primers (SEQ ID NO:10)18mer: 5′-ATGCCTACTCGCGAATTC-3′ 5′-GAACAGACCACCGGATCC-3′ 15mer:5′-CCTACTCGCGAATTC-3′ 5′-CAGACCACCGGATCC-3′ 10mer: 5′-CCTACTCGCG-3′5′-CAGACCACCG-3′  8mer: 5′-CCTACTCG-3′ 5′-CAGACCAC-3 Note: the 15-mer,10-mer and 8-mers are nested oligos of: SEQ ID NO:9 and SEQ ID NO:10,respectively.

Consequently, longer combinatorial sequence libraries would be possiblewith shorter primer sequences; in addition, shorter primer sections willreduce non-specific binding of target proteins to the ODN bead library.To study the primer length requirement for one-bead one-PCRamplification, a series of primers with varying lengths (8 mer, 10 merand 15 mer; see Table 1) were designed and synthesized to hybridize tothe 52 mer template ODN containing both 5′ and 3′ primer regions (ODN 2in Table 1) on the support beads. The PCR products of these primers weremonitored by 15% polyacrylamide gel electrophoresis. The PCR conditionswere optimized for each pair of primers of varying length. No detectableband was observed with the 8mer primers, even at the highestconcentration tested (data not shown). A weak band was detected with the10 mer primers, while a strong band was observed with 15 mer primers(data not shown). The fidelity in the Taq polymerase amplificationyielding the ODN products was confirmed by cloning and sequencing (datanot shown). These results suggest that ODNs with primer lengths of 10 ntor greater are required for efficient PCR amplification. In this study,15 mer primers were selected for the following studies.

Generation of a self-encoded S/S₂-ODN library. To introduction manycopies of a single, chemically pure S-ODN or S₂-ODN onto each bead, a“mix and separate” split synthesis method was used. A two-column DNAsynthesizer was used for constructing the library. The normal phosphatebackbone linkages were generated using standard phosphoramidite monomersvia oxidation in column 1, while the phosphorothioate orphosphorodithioate linkages were synthesized using standardphosphoramidite or thiophosphoramidite monomers via sulfurization incolumn 2, respectively. Two sequences of the same length are programmedfor each column and are designed such that the bases are different atevery equal position not only for diversifying base compositions butalso for coding a phosphate, phosphorothioate or phosphorodithioatelinkage. Thus, on an Expedite 8909 DNA synthesizer with dual columns,for example, onto column 1 a phosphoramidite (for example: dC) iscoupled to the bead and after completion of oxidation, the resultingproduct is nucleotide (dC) with a phosphotriester linkage. On column 2,a nucleoside phosphorothioate or phosphorodithioate is introduced with adifferent base (dT for example). The support beads from the two columnsare mixed and resplit and in the second cycle, additionalphosphoramidites or thiophosphoramidites are introduced, followed byoxidation and sulfurization reactions individually in columns 1 and 2.

After additional coupling steps and after final split/pool synthesis iscompleted, the end products comprise a combinatorial library of ODNswith varying thioate/dithioate or normal phosphate ester linkages atvarying positions along the ODN strand attached to the support. Eachbead contains a single chemical entity with a specified backbonemodification that is identified by the base. In the above example, anydC at position 1 of the sequence will be a 3′-phosphate while a dT atposition 1 would indicate that it contains a 3′-thiophosphate. Thisscheme was applied to synthesize a library of 4096 (2¹²) one-bead,one-S-ODN. This library included a 22-nucleotide combinatorial sequence(12 split/pool steps) flanked by 15 nucleotide defined primer regions atthe 5′ and 3′ ends (see Table 1). The 3′ ends of the sequences wereattached to the polystyrene beads. As noted above, the defined primersequences were incorporated to allow PCR amplification andidentification of the ODN sequence on the selected beads. Thus, thedownstream primers were first automatically synthesized in parallel onthe two columns. The S-ODN sequences of the combinatorial 22-mer segmenton each column were programmed for each column and were generated byintroducing a phosphorothioate linkage on every other base in column 2,following the “split and pool” approach. The identical upstream primersequences were then completed on both columns. As described below, asmaller S₂-ODN library was created in similar fashion.

Selection and sequencing of the S-ODN beads. Binding of thetranscription factor NF-κB p50/p50 homodimer and selection of specificbeads was demonstrated by first converting the single-stranded S-ODN todsDNA since the NF-κB transcription factor binds to DNA duplexes. Thesingle-stranded 52-mer S-ODN combinatorial library (typically 1-3 mg ofbeads) was converted to dsDNA by Klenow DNA polymerase I reaction in thepresence of DNA polymerase buffer, dNTP mixture and reverse primer.Therefore, one strand of the duplex potentially contained thiophosphatebackbone substitutions in the combinatorial library segment and theother complementary strand included an unmodified phosphate backboneODN. A duplex DNA library in which both strands contain S-ODNmodifications could also be generated using a Klenow reaction with nomore than three dNTP(α)S. Because the S-ODN strand attached to thesupport was chemically synthesized using phosphoramidite chemistry, eachthiophosphate is a mixture of R_(P) and S_(P) stereoisomers. The beadswere suspended in a diluted solution of NF-κB p50/p50 homodimer labeledwith the Alexa Fluor 488 dye at room temperature for 2 hrs.

Five positive beads from the S-ODN library were selected. Eachindividual bead was washed thoroughly with urea to remove the proteinand was directly used for the “one-bead, one-PCR” amplification usingthe 5′ and 3′ end primers described above. The PCR product was clonedand sequenced. Table 2 lists four of the S-ODN sequences obtained.

TABLE 2 S-ODNs/S₂-ODNs sequences identified from an NF-κB p50/p50protein screen^(a) Automated sequence Deduced S/S₂-ODN sequence S-ODNselection CTGTGAGTCGACTGATGACGGT CtGTGAGtCGACTgAtGaCGGtAGTTGAGTCGAAGGACCCATTT AGTTGAGtCGAaGgACCCAtTt CGTCAAGTCTCAGTTCCCATTTCGTcAAGtCtCaGTTCCCAtTt AGTCAAGTCGAAGTTCCACGGT AGTcAAGtCGAaGTTCCaCGGt(SEQ ID NOS:11-14) (SEQ ID NOS:15-18) S2-ODN selectionATGTAGCCAGCTAGTCTGTCAG ATGTAGCCAGCTAGTCTGTCAG^(b) CGCCAGCCAAAGGTGCTGTCAGCGCCAGCCAaAGGTGCTGTCAG CGCCCAGTGGCTAGTGAACCCC CGCCcAGTgGCTAGTgaACCCCATGTAGCCGAAGGTGGAACCCC ATGTAGCCgaAGGTGgaACCCC CGCCAGCCGAAGGTGGAACCCCCGCCAGCCgaAGGTGgaACCCC (SEQ ID NOS:19-23) (SEQ ID NOS:24-28) ^(a)Thelower case letter indicates a 3′-thioate linkage. The lower case boldletter indicates a 3′-dithioate linkage. ^(b)No 3′-dithioate linkagesare present in this strand.

Binding and Selection of combinatorial library of S₂-ODN beads. S₂-ODNsgenerally bind even more tightly to proteins than unsubstituted or S-ODNanalogues. Thus, it is significant that this S-ODN bead-basedcombinatorial selection method may be applied to dithiophosphatebackbone substitutions, since in vitro combinatorial selection is onlypossible for thiophosphate substituted ODNs with limited P-chirality. Todemonstrate how the present invention may overcome this limitation, asmall one-bead, one-S₂-ODN library was synthesized consisting of a poolof 32 (2⁵) sequences to allow further optimization of in vitro orbead-based S-ODN selected sequences. Chemical synthesis of S₂-ODN avoidsproblems created by a mixture of diastereoisomers of chemicallysynthesized S-ODN. The random region (5′-CGCCcAGTgaAGGTGgaACCCC-3′)(SEQID NO:28) in column 2 was identified as a S-ODN sequence derived from anin vitro combinatorial selection methodology that binds the NF-κBp50/p50 protein with high affinity (<20 nM) (All lower case lettersindicate enzymatically synthesized chiral 3′-thioate linkages). Theprogrammed combinatorial region sequence(5′-ATGTAGCCAGCTAGTCTGTCAG-3′)(SEQ ID NO:19) in column 1 was designedsuch that the bases at each 3′-dithioate position were different fromthe bases in column 2 at each equal position further allowing basesequence to identify backbone substitution. Thiophosphoramiditechemistry with sulfurization was used to generate 3′-dithioate linkages.Only the previous 3′-monothioate linkages were replaced with3′-dithioate linkages. The “split and pool” step followed most of thedithioate modifications. S₂-ODNs by selecting beads bindingfluorescently labeled NF-κB p50/p50 homodimer were also identified,followed by PCR amplification of 5 individually selected S₂-ODN beadsand cloning and sequencing of the PCR products. The sequences are alsolisted in Table 2.

The nucleic acid “aptamers” previously selected by incubating the target(protein, nucleic acid or small molecule) with the combinatorial libraryare then separated. The bound fractions were then amplified using PCRand subsequently reincubated with the target in a second round ofscreening. These iterations are repeated (often 10-20 cycles) until thelibrary is enhanced for sequences with high affinity for the target.Aptamers selected from combinatorial RNA and DNA libraries havegenerally had normal phosphate ester backbones, and so would generallybe unsuitable as drugs or diagnostics agents that are exposed to serumor cell supernatants because of their nuclease susceptibility. Rapiddegradation of natural ODNs used as antisense agents or aptamers bynucleases in serum or cells necessitates chemical modification of theODNs.

Among a large variety of modifications, S-ODN and S₂-ODN modificationsrender the agents more nuclease resistant. The first antisensetherapeutic drug uses a modified S-ODN (CIBA Vision, A NovartisCompany). The S₂-ODNs also show significant promise, however, the effectof substitution of more nuclease-resistant thiophosphates cannot bepredicted, since the sulfur substitution can lead to significantlydecreased (or increased) binding to a specific protein (Milligan, J. F.and Uhlenbeck, O. C. (1989) Determination of RNA-protein contacts usingthiophosphate substitutions. Biochemistry, 28, 2849-2290; and Yang, X.unpublished results) as well as structural perturbations and thus it isnot possible to predict the effect of backbone substitution on acombinatorially selected aptamer. Thus, if at all possible, selectionshould be carried out simultaneously for phosphate ester backbonesubstitution as well as the base sequence. Recently, an in vitrocombinatorial selection of thioaptamers from random orhigh-sequence-diversity libraries based on their tight binding to thetarget (e.g. a protein or nucleic acid) of interest was demonstrated byone or more of the present inventors.

Oligonucleotides possessing high fractional substitutions ofmonothio/dithioate internucleotide linkages appear to be “stickier”towards proteins than normal phosphate esters, and thereforethioaptamers with complete thiophosphate backbone substitutions appearto lose much of their specificity. This increased affinity is partly dueto the fact that the thioate groups only poorly coordinate hard cationssuch as sodium ions, and thus the thioaptamers serve as “bare” anionsand don't require any energy to strip away the neutralizing cations tobind to proteins. This observation of the increased affinity is of greatimportance to modified-ODN design as proteins recognize DNA at both thebases and phosphate esters. In previous studies, it was demonstratedthat binding of S₂—ODNs to a protein target requires only a limitednumber of phosphorodithioate linkages in a specific ODN sequence toachieve very high affinities (Gorenstein, D. G., et al., U.S. Pat. No.6,423,493, relvant portions incorporated herein by reference).

These results demonstrate that a split and pool synthesis may be used todevelop S-ODN, S₂—ODN and RNA libraries (which may also includeunmodified ODNs-whether single strand or duplex). In this procedure eachunique member of the combinatorial library was attached to a separatesupport bead. Targets that bind tightly to only a few of the potentiallymillions of different support beads were selected by binding the targetsto the beads and then identifying which beads have bound target bystaining and imaging techniques. The methodology of the presentinvention allowed the rapid screening and identification of aptamersthat bind to proteins, e.g., NF-κB, using a novel PCR-basedidentification tag of the selected bead.

These results demonstrate that the methodology can be applied to otherbackbone or base modifications that are compatible with templatescontaining these modifications. It is important that not only the S-ODNsbut even the S₂-ODNs are capable of acting as templates recognized byDNA polymerases for PCR amplification of selected S₂-ODN beads. Thisdemonstrates that nucleic acid analogues with phosphorodithioatelinkages can be used as a template in the nucleotidyltransferasereaction catalyzed by DNA polymerases. Likewise, polyamide nucleic acid(PNA) lacking the phosphate backbone may be recognized as a template forthe polymerase reaction.

In vitro selection of combinatorial libraries of S₂-ODNs is not possiblebecause dNTP (αS₂) is not a substrate for polymerases. The splitsynthesis, bead-based S₂-ODN library selection method of the presentinvention is the only method and/or library for identifying both optimalnumber and location of dithioate substitutions as well as base sequencesfor these S₂-ODN aptamers. Additionally, even for the thioate libraryselection, the in vitro methods involving iterative cycles of selectionisolation and reamplification of the bound members of the library by PCRamplification are very time consuming. In contrast, the single cycle ofsplit/pool synthesis, selection and identification of the presentinvention circumvents the need for the iterative cycles ofamplification, isolation and reamplification. The split pool bead-basedmethod and library of the present invention allows for theidentification of the positions of any 3′-monothioate/dithioatelinkages.

Although the beads were screened against a target protein labeled with afluorescent dye, the beads can also be screened directly against theunmodified transcription factor. The binding of the NF-κB to a specificsequence can be detected using a primary anti-NF-κB antibody, followedby a secondary antibody conjugated to a marker molecules includingfluorescein or rhodamine for fluorescence microscope (Yang, X.unpublished results).

To confirm the selection results, the S-ODN:5′-CtGTGAGtCGACTgAtGaCGGt-3′ (SEQ ID NO.: 15)(small letters representlocation of 3′-thioates), was independently synthesized on thenon-cleavable linker bead support, hybridized with its complementary ODNand then mixed again with the NF-κB p50/p50 protein labeled with theAlexa Fluor 488 dye. The fluorescence intensity of all of the beadsviewed under the fluorescence microscope was qualitatively similar tothe intensity of the selected bead containing this sequence within thecombinatorial library. These results demonstrate that the primer regionsdo not contribute to the binding of the NF-κB p50/50. Quantitativestudies on the affinities of the selected S-ODNs and S₂-ODN duplexes tothe protein along with selection from a large combinatorial library(10⁶˜10⁸) to NF-κB are in progress.

In earlier studies a thioaptamer clone obtained from an in vitrocombinatorial selection experiment (15 rounds of selection) bound toNF-κB p50/p50 with an apparent dissociation constant <5 nM(thiophosphate modification 5′ to each dA residue:5′-GGGGTTCCACCTTCACTGGGCG-3′•3′-CCCCAAGGTGGAAGTGACCCGC-5′)(SEQ ID NOS:29& 30). A chemically synthesized thioaptamer of the same sequence boundwith a dissociation constant of <20 nM. It should be noted, however,that each chemically synthesized thioaptamer consists of a diasteromericmixture containing 2^(n) different stereoisomers, where n is the numberof thiophosphates (2⁷=128 for the NF-κB p50/p50 selected thioaptamer).To determine the importance of the thiophosphate substitutions in thethioaptamer toward the NF-κB p50/p50 homodimer, a tight binding 15thround thioaptamer clone was synthesized by PCR with a nucleotide mixcontaining dATP instead of dATP(αS), and showed no binding of the normalphosphoryl backbone aptamer to NF-κB p50/p50 protein, supporting thecritical role played by the thiophosphates.

Phosphoramidite chemistry has been widely used for the synthesis ofS-ODNs because of its automation, high coupling efficiency and ease ofsite-specific thioate linkage incorporation. Synthesis of S-ODNs may becarried out by, e.g., forming an internucleoside phosphite linkagefollowed by sulfurization of the phosphite triester to aphosphorothioate. The resulting S-ODNs are a mixture ofdiastereoisomers, and consequently the diasteromeric S-ODN mixtures mayhave variable biochemical, biophysical and biological properties. Eachbead then contains a library of monothioate aptamers (a library oflibraries) since each bead contains the identical sequence and positionof thiophosphate substitution, but represents a mixture of diastereomersintroduced through the new monothiophosphate chiral centers.Stereocontrolled synthesis of a stereodefined S-ODN library may also beused to determine which is the best aptamer that binds to the protein,or may even be used to select a thioaptamer (or thioaptamer library)that has high affinity for the target protein or biomolecule. Thebinding data indicated that diastereoisomeric mixture libraries havegood selectivity and affinity, although not as high as purestereoisomers.

Another possible solution lies in the synthesis of modifications thatare achiral at phosphorus, such as the above S₂-ODN thioaptamer librarystudy. In addition dithioates appear to have greater “stickiness” toproteins than the thioates or unmodified ODN backbone.

The present invention may also be used to identify different nucleotidesequence(s) and/or to identify the backbone modification. S-ODN andS₂-ODN libraries were also created that differ only in the position ofphosphate or dithioate but not in its base sequence. It is known thatpositions of thiophosphates in a mixed backbone S-ODN sequence can bedetermined by reaction of the S-ODN with iodoethanol followed by basecatalyzed cleavage of the thiophosphate triester. The feasibility ofthis approach for identifying location of thioate linkages has beendemonstrated by the present inventors, and is often independent of basesequence.

The search for other split synthesis, bead-based combinatorial librariescontaining base modifications and hybrid backbones with phosphate ester,thioates, dithioates or potentially neutral methylphosphonates or evenpeptide nucleic acid chimeras with improved properties, such as enhancedbinding-affinity to a specific protein, increased biological stability,and improved cellular uptake, may be achieved by the split synthesiscombinatorial selection method described here.

By the split/pool method with two columns 2^(N) different members of thelibrary for N split/pool steps have been created. More columns (M) mayalso be used with the present invention to permit synthesis of M^(N)different beads with one unique thioaptamer sequence on each bead. Thelimit to the size of the combinatory library is the number of steps (N)and the number of columns (M) and of course the total number of beads,which generally is in the range of 10⁶ or more depending upon the sizeof the beads and synthesizer columns. Recently, aptamer beads on 15-20μm beads was achieved (Yang, unpublished) and thus a 40-fold increase inthe library size is possible. These results demonstrate that small beadsizes may be used effectively to produce more complex libraries atreduced cost and making more efficient use of reagents. The use of 15-20μm beads also demonstrates the scalability of the present invention.Finally, these results demonstrate that library sizes comparable tothose created by in vitro combinatorial selection methods by usingmixtures of phosphoramidites/thiophosphoramidites (up to 8 differentspecies) at selected positions in a given synthesis step may be created.The methodology of the invention may even be used to create a library oflibraries of beads, each bead containing a library of any complexity.Using the present invention a screener may easily create 10⁶ beads with10⁸ combinatorial library members on each bead—total diversity inprinciple is thus 10¹⁴, the same as in in vitro combinatorial selectionlibraries.

Sulfur substitution in aptamers alters the binding affinity and sequencethat is obtained by in vitro combinatorial selection methods.Post-selection phosphorothioate modifications of in vitrocombinatorially selected sequences can thus result in thioaptamers inwhich affinity cannot be reliably predicted. The simultaneous selectionfor both avoids this difficulty. The bead-based split synthesis,selection and PCR identification of combinatorial aptamer libraries nowprovides a means to combinatorially select both monothioate anddithioate variations on aptamers.

Flow cytometry sorting of thioaptamer bead-based library. The presentinventors also demonstrated the successful application of highthroughput/multi-color flow cytometry and bead sorting to screen aptamerbead libraries for those beads which bind to, e.g., a target protein.Modifications may be made to the flow cytometer to make it more amenableto bead identification and isolation. For example, bead fluorescence andforward scatter were the two parameters chosen for real-timecharacterization of each aptamer bead passing the first sort point of acustom-built flow cytometer/sorter. Other scanning and sortingparameters may be used to select, isolate, view, designate,characeterize, etc. the beads through a flow cytometer as will beapparent to those of skill in the art of cytometric analysis.

In operation, “positive” beads (contain thioaptamer-bound targetprotein, the target protein was fluorescent-labelled with Alexa 488 dye)were easily sorted from negative beads. Flow cytometry may be used toreplace, e.g., visual fluorescence microscope identification of beadscontaining bound target protein and the need to isolate the individual“positive” beads with a micromanipulator. The flow-sorted “positive”beads can then be subjected to, e.g., one-bead PCR to identify thethioaptamer that binds the target protein. The sorted “positive” beadsmay also be subjected to SELDI-MS analysis to confirm the identity ofthe bound protein (via molecular ion characterization). In cases wherethe “positive” bead's thioaptamer might have bound not only the targetprotein but other proteins in a sample, e.g., a secondary or eventertiary, etc. protein, SELDI-MS may be used to identify this eventthrough the detection of multiple molecular ions. These resultsdemonstrate that fluorescently protein-labeled beads are detected anddifferentiated from one another in a flow system in order to, e.g., sortout certain portions of the beads.

as the present inventors have demonstrated previously use of theone-bead, one-ODN:protein system using dual color sorting (U.S. patentapplication Ser. No. 10/272,509, filed Oct. 16, 2002, relevant portionsincorporated herein by reference). Briefly, the dsDNA κB consensussequences in the Igκ gene were immobilized onto 15-20 micron polystyrenemicrospheres. The DNA bound beads were then incubated with purifiedNF-κB p50 and p65 proteins, respectively. DNA transcription factorcomplexes were detected with primary antibodies specific for the NF-κBp50 and p65 proteins followed by an additional incubation with Alexa488-conjugated secondary antibody for NF-κB p50 and PE-conjugatedsecondary antibody for p65. The beads were viewed by fluorescentmicroscopy and then analyzed on the MCU's HiReCS system.

EXAMPLE II ELISA Based Thioaptamer Selection-Indirect ELDIA

Although the beads were screened against a target protein labeled with afluorescent dye, the beads can also been screened directly against thetranscription factor. The binding of the NF-κB to a specific sequencecan be detected using a primary anti-NF-κB antibody (Rabbit IgGantibody, Santa Cruz Biotechnology, Inc.) followed by a secondaryantibody conjugated with Alexa Fluor 488 (goat anti-rabbit IgG fromMolecular Probes). Next, several beads were selected for sequencing. Thesequencing result were as follows:

E008 Selected sequences 5′-CGCCAGCCGaAGGTGCTGTCAG-3′ (SEQ ID NO:31)5′-ATGTAGCCAaAGGTGgaACCCC -3′ (SEQ ID NO:32) 5′-CGCCcAGTgaAGGTGCTGTCAG-3′ (SEQ ID NO:33) 5′-CGCCcAGTAGCTAGTCTGTCAG -3′ (SEQ ID NO:34)

It was observed that the phosphorodithioate linkage (s) in the selectedabove sequences were different from those of the screening against thefluorescently labeled NF-κB p50. This result suggests that some of thebinding sites of NF-κB p50 protein may be preoccupied by fluorescentmolecules.

EXAMPLE III

Labeling of the ODN with Fluorescent Dyes

When synthesizing combinatorial libraries or specific thioaptamersequences on beads, one may also identify beads by attaching 2 or morefluorescent dyes to the ODN either at the 5′ or 3′ ends or internally byusing phosphoramidites with specific fluorophors attached. By using 1-3(or more) fluorophors at 2-3 or more different levels (individualnucleosides), it is possible to identify dozens or more of the sequencesor libraries by multicolor flow cytometry. (Each bead can thus beidentified by dye A, B and/or C at levels high, medium, low in variouscombinations: thus bead with A(hi), B(medium) and C(low) would be one ofdozens of different possible combinations.)

Thus it is possible to multiplex using flow cytometry or by randomlyplacing beads onto, e.g., the Texas tongue with hundreds or thousands ormore of different microwell holders, random assortment of thioaptamerbeads specific for binding different analytes. Alternatively, it ispossible to label fluorescently cell extracts with another dye and thenbind the protein(s) to the beads in conjunction with multicolor flow orsurface fluorescence, multiplex diagnostics chip or beads, as describedhereinbelow.

EXAMPLE IV

Fluorescent Tagging of Proteins Only with SELDI MS to Identify ProteinsExpressed Differentially

The thioaptamer combinatorial library may be used in conjunction withfluorescent tagging of proteins only and SELDI MS to identify proteinsdifferentially expressed in control vs. experiment. In this simpletwo-color assay, a combinatorial library (or a combinatorial library oflibraries) of beads is synthesized, each bead with a single thioaptamersequence (or a combinatorial library of thioaptamer sequences on eachbead). In this was we could create up to 10⁸ beads with a singlethioaptamer sequence on each bead.

Cell extracts of a sample are labeled fluorescently with a dye (cy3 forexample) and a control cell extract is labeled fluorescently withanother dye (cy5 for example). Both cell extracts are mixed together andallowed to bind to the bead thioaptamer library. Next, two (2) colorflow cytometry is used to compare cy3/cy5 color levels of each bead. Ifcy3/cy5 level differs from 1, then the bead may be captured. Todetermine which protein(s) have been bound to selected thioaptamer beadprotein determination technique, e.g., SELDI MS may be used tocharacterize the bound target further. SELDI MS may be used to determinewhich proteins have been bound to selected combinatory thioaptamerlibraries and also used with single bead PCR to identify which bead(s)in the combinatorial library have bound to protein(s). Pure thioaptamerbeads may be placed or spotted onto a chip or used in conjunction with,e.g., flow cytometry methods to bind the protein expresseddifferentially in a sample relative to control.

EXAMPLE V Fluorescent Tagging and SELDI to Identify Proteins ExpressedDifferentially

The thioaptamer combinatorial library may be used in conjunction withfluorescent tagging and SELDI to identify proteins differentiallyexpressed in control vs. experiment. Combinatorial libraries ofthioaptamer sequences on individual beads can be synthesized (forexample at position N1 on a split synthesis column 1 use 33% of A, G andT while on column 2 use C thiophosphoramidite to introduce either normalmixture of A/G/T phosphates or C dithiophosphate which can be identifiedby sequencing the N1 position. In this fashion we could create up to 10⁸beads with 10¹² combinatorial library members on each bead—totaldiversity in principle is 10²⁰ (of course the actually diversity is onlyabout 10¹⁴ at best).

Alternatively, the user may create only 100 different split synthesisbeads each with up to 10¹² combinatorial library members on the beads.By using, e.g., 3 or more fluorophors, attached at various levels viaphosphoramidite chemistry to the ODN it is possible to use flowcytometry to identify each bead library. For example, sample cellextracts may be labelled fluorescently with one dye (cy3 for example)and control cell extracts may be labelled fluorescently with another dye(cy5 for example). Use of five (5) color flow cytometry may also be usedto compare cy3/cy5 color levels of each bead library that is identifiedby covalently attached flurophors to one or more of the thioaptamer(s).If cy3/cy5 level differs from 1, then sort the beads by the fluorophoretags for each non-unitary cy3/cy5 levels. As with the previous Example,SELDI MS may be used to determine which proteins have been bound toselected combinatory thioaptamer libraries and also used with singlebead PCR to identify which bead(s) in the combinatorial library havebound to protein(s). In one embodiment of the present invention it ispossible to incorporate a modified nt phosphoramidite (at the C-5pyrimidine position for example) in the combinatorial library sequencepositions to create a tag for the libraries and thus create 100libraries in one split/pool synthesis. Alternatively, it is possible touse photoactivated crosslinkers to attach the protein to the thioaptamer(e.g., BrU on a single strand). Proteolysis of protein(s) may be used inconjunction with MS to identify the bound peptides and/or proteins. Itis also possible, as described hereinabove, to use single-bead PCR toidentify which bead(s) from the combinatorial library have bound to theprotein(s). Since the BrU is on only 1 strand of the thioaptamer, theother can be sequenced by PCR. To identify the exact thioaptamersequence that bound to the protein, then a four (4) column split/poolsynthesizer may be needed. Alternatively, it is possible to spot purethioaptamer or library onto chip and use this spot to bind thedifferentially expressed protein under the sample relative to control.

EXAMPLE VI Synthesized a Monothio RNA Library

The present inventors have also successfully synthesized a monothio RNAlibrary (2¹⁵=32768). Standard phosphoramidite (DNA and RNA) chemistrywas used for the monothio RNA library. A 0.5 M 1H-tetrazole inacetonitrile was used as DNA activator. A 0.5 M solution of DCI(dicyanoimidazole) in acetonitrile was used as RNA activator. Thelibraries were prepared on a 1 μmole scale of polystyrene beads (66-70μm). The downstream and upstream primers, 5′-d(GGATCCGGTGGTCTG)-3′ (SEQID NO:35) and 5′-d(CCTACTCGCGAATTC)-3′ (SEQ ID NO:36) were synthesizedin parallel on a two-column DNA synthesizer (Expedite 8909, AppliedBiosystems). Following the 5′-primer, the sequences programmed on thesynthesizer for the combinatorial mono RNA library were5′-r(GA*UC*CU*GA*AA*CU*GU*UU*UA*AG*GU*UG*GC*CG*AU*C)-3′ (SEQ ID NO:37)on column 1 and 5′-r(cU*aG*gA*cU*uG*gC*aC*aA*cC*gU*cA*cA*cU*gC*uA*u)-3′(SEQ ID NO:38) on column 2. The 3′-primer sequence completed the 61-merprogrammed on the synthesizer.

A “split and pool” occurred at each position indicated by an asterisk inorder to synthesize the combinatorial region for the monothio RNA. Thelower case letter indicates a 3′-thioate linkage, the upper case letterindicates a 3′-phosphate linkage. The coupling yield was typicallyupwards of 98.5% as determined by the dimethoxytrityl cation assay.Sulfurization chemistry utilized the Beaucage reagent. The fullyprotected monothio RNA combinatorial library with the non-cleavablelinker beads were treated with 4 ml of a mixture of 3:1 (v/v) (28%) NH₃:EtOH at 39° C. for 21 hrs. The beads were centrifuged, the supernatantwas removed and the solid support was washed with double-distilledwater. After lyophilization the solid support was treated with 2 ml oftriethylamine trihydrofluoride (TEA-3HF) for 20 hrs at room temperature.Again, the beads were centrifuged, the supernatant was removed and thesolid support was washed with double-distilled water.

Column 1: (SEQ ID NO:39) 5′-CCTACTCGCGAATTC-GA*UC*CU*GA*AA*CU*GU*UU*UA*AG*GU*UG*GC*CG*AU*C-GGATCCGGTGGTCTG-Linker-3′: Phosphate Column 2: (SEQ ID NO:40)5′-CCTACTCGCGAATTC- CU*AG*GA*CU*UG*GC*AC*AA*CC*GU*CA*CA*CU*GC*UA*G-GGATCCGGTGGTCTG-Linker-3′: MonothioRNA

EXAMPLE VII NMR Spectra of XBY-5 and XBY-15

The NOESY, DQCOSY and TOCSY spectra of XBY-2, XBY-6, XBY-5 (200 OD) andXBY-15 (90 OD) have been acquired. NMR structures for XBY-2 and XBY-6have been determined and shown to differ from the structure of theparent duplex sequence without any dithioate substitutions (Volk, etal., in press). The structures of the other two thioaptamers are beingdetermined.

XBY-5: 5′-CC AGGAGAT _(S2) T _(S2)CCA C-3′ (SEQ ID NO:41) 3′-GG_(S2) TCCTCTA A GG_(S2) TG-5′ (SEQ ID NO:42) XBY-15: 5′-CC A_(S2)G_(S2)GAGAT_(S2)T_(S2)CCAC-3′ (SEQ ID NO:43) 3′-GGT_(S2) C _(S2) CT_(S2) CTA AGGTG-5′ (SEQ ID NO:44)

EXAMPLE VIII High Quality of One-Bead, One-ODN Library Ligation Reaction

The present inventors demonstrated that they could construct highquality one-bead one-oligo libraries by join two pieces of DNA based onligation reaction or highly active phosphorothioate towards 5′-iodogroups on the ODN. Standard phosphoramidite chemistry was used forsynthesis of 5′ monophosphate ODN(5′-P(o)CCAGGAGATTCCAC-GGATCCGGTGGTCTGT-bead) (SEQ ID NO:45). The fullyprotected ODN with the non-cleavable linker beads were treated withconcentrated ammonia at 37° C. for 21 hours to remove the protectinggroups while allowing the ODN to remain attached to the beads. Aselected single bead was mixed with the following components: 3 μl of 40μM 15 mer oligonucleotide (5′-CCTACTCGCGAATTC-3′ (SEQ ID NO:36), 3 μl of10× ligation buffer, 3 μl of DMSO, 2 μl of T4 RNA ligase and 19 μl ofddH₂O. The reaction was performed at 5° C. for 17 hrs. The supernatantwas removed carefully and washed with water. The single bead wasperformed PCR reaction at established conditions. The PCR products wereanalyzed on a 15% native polyacrylamide gel. The PCR product was clonedusing the TA Cloning procedure (Invitrogen) and sequenced on an ABIPrism 310 Genetic Analyzer (Applied Biosystems). The desired sequence(5′-CCTACTCGCGAATTC-P(o)CCAGGAGATTCCAC-GGATCCGGTGGTCTGT-bead) (SEQ IDNO:46), was obtained.

To demonstrate the ligation reaction, a simple ODN was ligated to asingle bead of a one-bead, one-ODN library, namely:

The ligation reaction was confirmed by one-bead PCR reaction and cloningand sequencing. These results show that the additional nucleic acidsequences may be added to one or more of the beads of a one-bead,one-ODN library with high quality and efficiency while maintaining theintegrity of the library. The ligation reaction allows longer randomregions of aptamers to be synthesized on the beads with higher yieldsince a primer region does not have to be stepwise synthesized onto thebead sequence.

EXAMPLE IX

Separation of Synthetic Oligonucleotide Dithioates fromMonothiophosphate Impurities by Anion-Exchange Chromatography on a MonoQ Column

A method using a strong anion-exchange liquid chromatography column,Mono Q, has been developed for high resolution analysis and purificationof oligonucleotide dithioates, which were synthesized by an automated,solid-phase, phosphorothioamidite chemistry. High-resolution separationof oligonucleotide phosphorodithioates from monothiophosphate impuritieswas obtained. High-resolution separation was also demonstrated at pH 8.The separation of oligonucleotide dithioates was found to be linearlydependent on the number of sulfurs for the same sequence length.Thiocyanate, SCN⁻, as eluting anion, can be used to purifyoligonucleotides containing a high percentage of phosphorodithioatelinkages in lower salt concentration, and provide better separation thanthat of chloride as eluting anion.

Synthesis of oligomers. The following oligomers were synthesized forthis study:

New Scramble: (SEQ ID NO:47) 5′-CCA GT_(S2)GA CT_(S2)CA GT_(S2)G-3′ (SEQID NO:48) 3′-GGT_(S2)CA CT_(S2)GA GT_(S2)CA C-5′ 5′-amino-xby6: (SEQ IDNO:49) 5′-H₂NC₁₂H₂₄—O₃P—O—CCAGG A GA T_(S2)T_(S2)CCA C-3′ (SEQ ID NO:50)3′-GGT_(S2)CCT_(S2)CT_(S2)A A GGT_(S2)G-5′ 5′-fluorescein-xby6: (SEQ IDNO:51) 5′-C₆H₁₂—O₃P—O—CCA GGA GA T_(S2)T_(S2)CCA C-3′, (SEQ ID NO:52)3′-GGT_(S2)CCT_(S2)CT_(S2)A A GGT_(S2)G-5′ XBY-6 and IgkB-22 on Beadsfor: (SEQ ID NO:53) 5′-AGTTGAGGGGACTTTCCCAGGCTT-bead (IgkB) (SEQ IDNO:54) 3′-TCAACTCCCCTGAAAGGGTCCG-5′ (SEQ ID NO:55) 5′-CC AGG AGAT_(S2)T_(S2)CC AC-linker-bead (XBY-6) (SEQ ID NO:56)3′-GG_(S2)TCC_(S2)TC_(S2)T A A GG_(S2)TG-5′

XBY20-26 for EMSA Competition Assay XBY-20: dithioP50-1: 5′-CGC CC_(S2)AGTG_(S2)A_(S2)AG GTG G_(S2)A_(S2)A (SEQ ID NO:57) CCCC-3′ dithioP50-1c:5′-GGG GTT CC_(S2)A C CTT C_(S2)AC TGG (SEQ ID NO:58) GCG-3′ XBY-21:dithioP50-2: 5′-CGC CC_(S2)A GTG_(S2)AAG GTG GA_(S2)A (SEQ ID NO:59)CCCC-3′ dithioP50-1c: 5′-GGG GTT CC_(S2)A C CTT C_(S2)AC TGG (SEQ IDNO:60) GCG-3′ XBY-22: dithioP50-3: 5′-CGC CC_(S2)A GTGAAG GTG GA_(S2)A(SEQ ID NO:61) CCCC-3′ dithioP50-1c: 5′-GGG GTT CC_(S2)A C CTT C_(S2)ACTGG (SEQ ID NO:62) GCG-3′ XBY-23: dithioP50-1: 5′-CGC CC_(S2)AGTG_(S2)A_(S2)AG GTG G_(S2)A_(S2)A (SEQ ID NO:63) CCCC-3′phosphateP50-1c: 5′-GGG GTT CCA C CTT C AC TGG GCG-3′ (SEQ ID NO:64)XBY-24: dithioP50-2: 5′-CGC CC_(S2)A GTG_(S2)AAG GTG GA_(S2)A (SEQ IDNO:65) CCCC-3′ phosphateP50-1c: 5′-GGG GTT CCA C CTT C AC TGG GCG-3′(SEQ ID NO:66) XBY-25: dithioP50-3: 5′-CGC CC_(S2)A GTGAAG GTG GA_(S2)A(SEQ ID NO:67) CCCC-3′ phosphateP50-1c: 5′-GGG GTT CCA C CTT C AC TGGGCG-3′ (SEQ ID NO:68) XBY-26: PhosphateP50-1: 5′-CGC CCA GTGAAG GTG GAACCCC-3′ (SEQ ID NO:69) dithioP50-1c: 5′-GGG GTT CC_(S2)A C CTT C_(S2)ACTGG (SEQ ID NO:70) GCG-3′

It was found that the XBY20-26 ODN does not compete as well as theselected oligo (monothio selected) with the recombinant NF-κB p50. Thechemically synthesized selected oligo (2) was the best so far.

Example X

Dispersal and imaging of fluorescent-labelled microbeads within athixotropic gel 50 uL, 100 uL and 150 uL of fluorescently labelled 66micron beads, that includes approximately 14,000, 30,000 and 42,000beads, respectively, were each added to approximately 5 mL of 15%PAGE-GEL solution. Each bead-gel solution was vortexed and then 5 uL, 10uL and 15 uL of TEMED (tetramethylene diamine) gel polymerizationcatalyst was added to each vortexed solution, respectively, and thebead-gel was immediately loaded onto a Bio-Rad mini-gel system. The 7×10cm gels, of 0.7 mm thickness, were then viewed on a Perkin-ElmerProEXpress 2D gel imager. FIG. 8 shows the image of a gel treated asabove, but without the addition of the fluorescent beads, thus acting asthe control image. FIGS. 9-11 show the images of the bead-gels having14,000, 30,000 and 42,000 beads. The beads are relatively uniformlydispersed in all three images. FIGS. 8-11 demonstrate feasibility ofuniformly dispersing the aptamer beads within a thixotropic 2D gelmatrix and of imaging the bead-gels via detection of the fluorescence ofthe labelled beads, providing the signal and image location that can beused by a commercially available robotic spot picker to address beads ofinterest and both chemically and mechanically manipulate such beads.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

While this invention has been described in reference to illustrativeembodiments, this description is not intended to be construed in alimiting sense. Various modifications and combinations of theillustrative embodiments, as well as other embodiments of the invention,will be apparent to persons skilled in the art upon reference to thedescription.

1-30. (canceled)
 30. A method for aptamer selection comprising the stepsof: dispersing a one-aptamer, one-bead combinatorial bead library into atwo-dimensional matrix; scanning for aptamer beads that generate adetectable signal from interaction between the one or more aptamer beadsand a target; and picking one or more aptamer beads based on thedetectable signal from within the matrix.
 31. The method of claim 30,further comprising the step of extracting the target from the aptamerbead.
 32. The method of claim 30, further comprising the step ofidentifying the target by mass spectrometry after liquid chromatography.33. The method of claim 30, wherein the one-aptamer, one-beadcombinatorial bead library is dispered within the matrix by molecularprinting.
 34. The method of claim 30, wherein the one-aptamer, one-beadcombinatorial bead library is dispered within the matrix by molecularprinting is via an inkjet printer.
 35. The method of claim 30, whereinthe matrix comprises a gel, a polymer, a thixotropic agent, a glass or asilicon matrix.
 36. The method of claim 30, further comprising the stepof separating the target into one or more peptides prior to separationby liquid chromatography.
 37. The method of claim 30, wherein the stepsof identifying the target by mass spectrometry is preceded by the stepsof extracting and separating the proteins by liquid chromatography. 38.The method of claim 30, wherein the steps of identifying the targetusing mass spectrometry comprises matrix assisted laser desorptionionization (MALDI) mass spectrometry.
 39. The method of claim 30,wherein the library comprises an S-ODN library.
 40. The method of claim30, wherein the library comprises an S₂-ODN library.
 41. The method ofclaim 30, wherein each of the aptamers is further modified to comprise acolorimetric agent.
 42. The method of claim 30, wherein each of theaptamers further comprises one or more bases that are attached to afluorophor.
 43. The method of claim 30, wherein each of the aptamersfurther comprises one or more fluorophors attached to the 5′ end, the 3′end or internally within the aptamers.
 44. The method of claim 30,further comprising the complementary strand to the aptamer.
 45. Themethod of claim 30, wherein the aptamer is defined further as athioaptamer.
 46. The method of claim 30, wherein the aptamer comprises athioaptamer wherein one or more but less than all of the linkagescomprising one or more of the following: rATP(αS), rUTP(αS), rGTP(αS),rCTP(αS), rATP(αS₂), rUTP(αS₂), rGTP(αS₂), rCTP(αS₂), rATP(αS),dTTP(αS), dGTP(αS), dCTP(αS), dATP(αS₂), dTTP(αS₂), dGTP(αS₂) anddCTP(αS₂).
 47. The method of claim 30, wherein the target is labeledwith an enzyme, a dye, a radioisotope, an electron dense particle, amagnetic particle, a fluorescent agent, an antibody, a magnetic particleor a chromophore.
 48. The method of claim 30, wherein the target isdetectable with an enzyme, a radioisotope, an electron dense particle, amagnetic particle, a fluorescent agent, an antibody, a magnetic particleor a chromophore.
 49. The method of claim 30, wherein the aptamer beadis further processed to remove the target bound to the aptamer bead. 50.The method of claim 30, wherein the aptamer bead is acquired by ascanning robotic head and the target is extracted from the aptamer beadin situ.
 51. The method of claim 30, aptamer bead is acquired by ascanning robotic head and the target is extracted from the aptamer beadin situ by proteolysis and transferred to the inlet of an LC-MS or anLC-MS/MS.
 52. The method of claim 30, wherein the aptamer bead isacquired by a scanning robotic head and the target is extracted from theaptamer bead in situ for MALDI-MS analysis, wherein the MALDI-MSanalysis is selected from the group consisting of MALDI-TOF/MS,MALDI-TOF/TOF-MS and MALDI-Q-TOF-MS.
 53. The method of claim 30, whereinthe aptamer bead is acquired by a scanning robotic head and the targetis extracted from the aptamer bead in situ for LC-MS analysis.
 54. Themethod of claim 30, wherein the aptamer bead is acquired by a scanningrobotic head and the target is extracted from the aptamer bead in situfor MALDI-MS analysis.
 55. The method of claim 30, wherein the aptamerbead is acquired by a scanning robotic head and the target is extractedfrom the aptamer bead in situ for MALDI-MS analysis by SELDI ionization.56. The method of claim 30, wherein the aptamer bead is furtherprocessed to remove the target bound to the aptamer bead and analyzingthe target by MS, MS/MS, MALDI-TOF, MALDI-TOF-MS, direct sequencing. 57.The method of claim 56, wherein the MALDI ionization step is a SELDIionization.
 58. The method of claim 30, wherein the aptamer bead isfurther processed to remove the target bound to the aptamer bead andanalyzing the target by binding a second detectable label to the target.59. The method of claim 30, wherein the matrix comprises apolyacrylamide gel, an alkyd resin or a silica-lipid.
 60. The method ofclaim 30, wherein picking the beads is selected from picking manually,semi-manually or non-manually.
 61. The method of claim 30, wherein thetarget is selected from peptides, proteins, nucleic acids,carbohydrates, lipids or combinations thereof. 62-81. (canceled)